This invention relates to an apparatus that generates a thermal gradient, particularly on a wafer. This invention also relates to methods of using the thermal gradient in molecular interactions, particularly for characterizing interactions involving biological macromolecules.
The stability and interactions of biological macromolecules are determined by a number of forces including, for example, ionic forces, van der Waals forces, and hydrogen bonds. Hydrogen bonds are known to be fairly weak and heat-labile forces in biological macromolecules. Small changes in the environment, in particular the temperature of the biological macromolecules, can alter the intramolecular and/or intermolecular hydrogen bonding of the macromolecules. Biological macromolecules, thus, can be sensitive to even small fluctuations in the environment.
Hybridization between nucleic acid molecules requires successful formation of hydrogen bonds between complementary nucleic acid molecules. Because hybridization relies on weak, heat-labile hydrogen bonds, hybridization is an exquisitely temperature sensitive process. Small fluctuations in the temperature and/or in the sequence of the nucleic acids can affect hybridization between complementary nucleic acid molecules.
Currently, information derived from hybridizations conducted on deoxyribonucleic acid (DNA) chips is stimulating advances in drug development, gene discovery, gene therapy, gene expression, genetic counseling and plant biotechnology. A DNA chip is a rigid flat surface, typically glass or silicon, with short chains of related nucleic acids spotted in rows and columns on it. As an example, hybridization between a fluorescently labeled single stranded nucleic acid molecule and nucleic acid molecules at specific locations on the chip can be detected and analyzed by computer-based instrumentation.
Among the technologies for creating DNA chips are photolithography, xe2x80x9cor-chipxe2x80x9d synthesis, piezoelectric printing and direct printing. Chip dimensions, the number of sites of DNA deposition (sometimes termed xe2x80x9caddressesxe2x80x9d) per chip and the width of the DNA spot per xe2x80x9caddressxe2x80x9d are dependent upon the technologies employed for deposition. The most commonly used technologies presently produce spots with diameter of 50-300 micrometers (xcexcm). Photolithography produces spots that can have diameters as small as 1 xcexcm. Technologies for making such chips are described, for example, in U.S. Pat. No. 5,925,525 to Fodor et al., U.S. Pat. No. 5,919,523 to Sundberg et al., U.S. Pat. No. 5,837,832 to Chee et al. and U.S. Pat. No. 5,744,305 to Fodor et al. which are incorporated herein by reference.
Hybridization to nucleic acids on DNA chips can be monitored, for example, by fluorescence optics, by radioisotope detection, and mass spectrometry. The most widely-used method for detection of hybridization employs fluorescence-labeled DNA, and a computerized system featuring a confocal fluorescence microscope (or an epifluorescence microscope), a movable microscope stage, and DNA detection software. Technical characteristics of these microscope systems are described in U.S. Pat. No. 5,293,563 to Ohta, U.S. Pat. No. 5,459,325 to Hueton et al. and U.S. Pat. No. 5,552,928 to Furuhashi et al. which are incorporated herein by reference. Further descriptions of imaging fluorescently labeled immobilized biomolecules and analysis of the images are set forth in U.S. Pat. No. 5,874,219 to Rava et al., U.S. Pat. No. 5,871,628 to Dabiri et al., U.S. Pat. No. 5,834,758 to Trulson et al., U.S. Pat. No. 5,631,734 to Stern et al., U.S. Pat. No. 5,578,832 to Trulson et al., U.S. Pat. No. 5,552,322 to Nemoto et al. and U. S. Pat. No. 5,556,539 to Mita et al. which are incorporated herein by reference.
Currently, manipulations performed with DNA chips are limited to protocols in which all of the samples on a chip are at about the same temperature. Simple and inexpensive methods of creating temperature differentials on DNA chips would greatly expand the repertoire of procedures available that can be performed on a DNA chip.
In a first aspect, the invention pertains to an apparatus. The apparatus includes a semiconducting wafer and two electrical connectors that are adjacent to each other on the wafer. Each of the connectors are attached to the wafer at an attachment site on the wafer with a gap disposed between the two attachment sites. A power source is connected to the wafer through the two electrical connectors.
In a further aspect, the invention pertains to a method of generating a temperature gradient. The method includes attaching two electrical connectors to a semiconducting wafer, wherein each of the connectors are adjacent to each other and attached to the wafer at an attachment site with a gap disposed between the attachment sites. The method also includes connecting a power source to the wafer through the two electric connectors.
In another aspect, the invention pertains to a method of analyzing biological macromolecules. The method includes establishing a temperature gradient on a semiconducting wafer having a stratum disposed thereupon. The stratum has one or more samples that include biological macromolecules in thermal contact with the temperature gradient. The wafer has two electrical connectors connected to opposite poles of an electrical power source. The method also includes evaluating the samples to determine thermal stability of complexes formed with the biological macromolecules in the samples. The samples are evaluated by measuring a property of the sample.
In a further aspect, the invention pertains to a method of conducting nucleic acid hybridization. The method includes establishing a temperature gradient on a stratum disposed on a semiconducting wafer. One or more samples including nucleic acid molecules are disposed on the stratum that is in thermal contact with the temperature gradient. Two electrical connectors are connected to the wafer and to opposite poles of an electrical power source. The method also includes performing a hybridization protocol on the one or more samples to determine temperature effect based on the gradient.
In yet another aspect, the invention pertains to a method of assessing binding complex interactions. The method includes establishing a temperature gradient on a semiconducting wafer having a stratum disposed thereupon. The stratum has one or more samples, each sample including one or more members of a binding complex in thermal contact with the temperature gradient. The wafer has two electrical connectors connected to opposite poles of an electrical power source. The method also includes evaluating the samples to determine thermal stability of the binding complex on the stratum.
In a further aspect, the invention pertains to a method of generating a temperature gradient on a stratum. The method includes placing the stratum in thermal contact on a surface having a temperature gradient wherein the stratum has low thermal conductivity.